Molecular Mechanisms of Cellular Signaling by the Proton Pumping Vacuolar ATPase.

PROJECT SUMMARY Altered pH, metabolic homeostasis and autophagy are hallmarks of cancer, neurodegenerative and infectious diseases. These fundamental processes are linked by a ubiquitous and essential ATP-dependent proton pump called the vacuolar ATPase (V-ATPase). The V-ATPase is a large, multisubunit, membrane-integral molecular machine whose canonical function is to acidify intracellular organelles in all eukaryotic cells according to their functional requirements. More recently, the V-ATPase has emerged in crucial non-canonical signaling roles including, but not limited to metabolic homeostasis and vesicular trafficking. This research program aims to discover the molecular mechanisms that drive V-ATPase’s ability to both acidify organelles and interact with cellular factors to mediate signaling pathways. The current hypothesis is that the ability of V- ATPase to multitask in these interdependent functions involves changes in V-ATPase subunit composition and conformations that are linked to the enzyme’s reaction cycle. To address these questions, (1) The complete reaction cycle of the V-ATPase will be delineated using cryo-electron microscopy (cryo-EM) coupled with in vitro biochemical assays (e.g., ATPase and proton pumping activity assays) and established yeast phenotypes for V-ATPase activity. (2) On early endosomes, the V-ATPase acts as a pH-sensor to recruit a GTPase (Arf-6) and its GEF (Cytohesin-2) for vesicular trafficking of substances to the lysosome. The molecular mechanism of this process will be dissected using protein-protein interaction assays, cryo-EM, and endocytosis/trafficking of albumin in kidney proximal tubule cells. (3) On lysosomes, the V-ATPase mediates metabolic signaling by forming distinct supercomplexes with members of the mechanistic target of rapamycin complex 1 (mTORC1) or AMP-dependent kinase (AMPK) pathways. It is hypothesized that the V-ATPase, bound to a GEF called Ragulator acts as a common molecular switch to promote either mTORC1 or AMPK signaling. The mechanism of V-ATPase-mediated metabolic signaling will be elucidated using protein-protein interaction studies, in vitro reconstitutions, affinity purification mass spectrometry, cryo-EM, and in-cell phosphorylation assays for mTORC1/AMPK activity. The feasibility of this work is supported by the candidate’s expertise in the field as well as robust preliminary experiments showing cryo-EM of V-ATPase (pertinent to goals 1, 2 and 3) and interaction of V-ATPase with Cytohesin-2 (goal 2) and Ragulator (goal 3). Collaborations with leaders in molecular dynamics and mass spectrometry further support these studies. Overall, this research program will not only improve our understanding of V-ATPase mechanics but also reveal key details of cellular pathways implicated in disease conditions.